Poor or Non-Linear Standard Curve (ELISA (Sigma Guide))

Question

Accepted Answer

Standard curve shows irregular shape, poor linearity, or inconsistent serial dilution response. R-squared value below 0.95 or curve does not follow expected sigmoidal or linear pattern across dynamic range. Common causes: 1) Pipetting errors or calculation mistakes in preparing serial dilution series 2) Poor or variable adsorption of coating reagents to plate surface 3) Capture antibody did not properly bind to plate due to incorrect coating buffer or presence of interfering proteins 4) Reagents poorly mixed before dispensing into wells 5) Inhomogeneous samples or improper ELISA plate selection Solutions: 1) Verify pipetting technique and dilution calculations; use calibrated pipettes; prepare fresh dilution series 2) Use proper coating buffer: PBS pH 7.4 or carbonate-bicarbonate buffer pH 9.6; extend coating incubation time if needed 3) Dilute capture antibody in PBS without other proteins; verify proper high-binding ELISA plate is used 4) Thoroughly mix reagents before use; vortex or invert multiple times 5) Check sample homogeneity; consider different plate types (high-binding vs medium-binding); use appropriate plates for fluorescence detection if applicable